U.S. patent number 5,218,089 [Application Number 07/799,421] was granted by the patent office on 1993-06-08 for retro-inverso analogues of thymopentin and the method for their synthesis.
This patent grant is currently assigned to Sclavo S.p.A.. Invention is credited to Sabina Mariotti, Luciano Nencioni, Alessandro Sisto, Antonio S. Verdini, Luigi Villa.
United States Patent |
5,218,089 |
Mariotti , et al. |
June 8, 1993 |
Retro-inverso analogues of thymopentin and the method for their
synthesis
Abstract
New analogues of thymopentin (TP5) and of its tetrapeptide
fragment (TP5.sup.1-4) containing two non-contiguous retro-inverted
bonds in the peptide chain are described which are of the general
formula (I) ##STR1## where R is hydrogen or an acyl radical, and
R.sup.1 is an --OR.sup.2 group or an ##STR2## group where R.sup.2
is a hydrogen atom or a hydrocarbon radical, and R.sup.3 is a
hydrogen atom or a hydroxyl group, and the corresponding
pharmaceutically acceptable salts of acid or basic addition,
possess immunomodulating activity.
Inventors: |
Mariotti; Sabina (Fara Sabina,
IT), Sisto; Alessandro (Rome, IT),
Nencioni; Luciano (Poggibonsi, IT), Villa; Luigi
(Florence, IT), Verdini; Antonio S. (Monterotondo,
IT) |
Assignee: |
Sclavo S.p.A. (Siena,
IT)
|
Family
ID: |
27273342 |
Appl.
No.: |
07/799,421 |
Filed: |
November 26, 1991 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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454282 |
Dec 21, 1989 |
5091510 |
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Foreign Application Priority Data
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Dec 23, 1988 [IT] |
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23099 A/88 |
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Current U.S.
Class: |
530/333; 530/323;
530/330; 530/332; 530/334 |
Current CPC
Class: |
C07K
5/0212 (20130101); C07K 7/02 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
7/00 (20060101); C07K 7/02 (20060101); C07K
5/00 (20060101); C07K 5/02 (20060101); A61K
38/00 (20060101); A61K 037/02 (); C07K 005/00 ();
C07K 007/00 (); C07K 015/00 () |
Field of
Search: |
;514/11,17
;530/323,330,332,333,334 |
References Cited
[Referenced By]
U.S. Patent Documents
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4505853 |
March 1985 |
Goldstein et al. |
4547489 |
October 1985 |
Goldstein et al. |
4629723 |
December 1986 |
Goldstein et al. |
5091510 |
February 1992 |
Mariotti et al. |
5116947 |
May 1992 |
Pinori et al. |
|
Foreign Patent Documents
Other References
Denes et al., Chemical Abstracts, vol. 107, 1987, p. 60. Abst No:
211960d..
|
Primary Examiner: Lee; Lester L.
Assistant Examiner: Davenport; A. M.
Attorney, Agent or Firm: Hedman, Gibson & Costigan
Parent Case Text
This is a divisional, of application Ser. No. 07/454,282, filed
Dec. 21, 1989 and now U.S. Pat. No. 5,091,510.
Claims
We claim:
1. A process for preparing a compound of formula (I) ##STR20##
where R is hydrogen or an acyl radical, and R.sup.1 is an
--OP.sup.2 group or an ##STR21## group where R.sup.2 is a hydrogen
atom or a C.sub.1 -C.sub.6 linear or branched chain alkyl radical,
a C.sub.3 -C.sub.6 linear or branched chain alkenyl or alkynyl
radical, or a C.sub.7 -C.sub.12 aryl-alkyl or alkyl-aryl radical,
and R.sup.3 is a hydrogen atom or a hydroxyl group,
a) by condensing a compound of formula (II) ##STR22## where P.sup.L
indicates a protecting group for the side chain amino function, and
P.sup.G indicates a suitable protecting group for the guanidino
function, with a compound of formula (III) ##STR23## where R.sup.4
is a --OR.sup.2 group in which R.sup.2 is defined as above, an --OP
group where P is a protecting group for the carboxyl function, or a
group of formula (IV) ##STR24## where P is as heretofore defined,
and R.sup.5 is a hydrogen atom or --OP.sup.I group where P.sup.I is
a protecting group for the hydroxyl function of the tyrosine,
wherein the compound of formula (III) is prepared by condensing, in
the presence of a suitable sylanizing agent, an amide of formula
(X), said compound (X) being obtained from D-aspartic acid,
##STR25## where P is a protecting group for the carbonyl function,
with a compound of formula (XI) ##STR26## where R.sup.4 is a group
of formula (IV), then converting the amide to primary amine with
TIB, said compound of formula (XI) being prepared by condensing, in
presence of a suitable sylanizing agent, the compound of formula
(XIV) ##STR27## where R.sup.6 is --CH(CH.sub.3)CH.sub.3 with a
compound of formula (XIII) ##STR28## where P and R.sup.5 are
defined as above. b) followed by conversion of the terminal amino
group of the obtained intermediate of formula (V) ##STR29## into a
primary amino group by treatment with
1,1-bis-trifluoroacetoxy-iodobenzene to give an intermediate of
formula (VI) ##STR30## c) followed by possible acylation of the
terminal amino function formed, and the removal of the protecting
groups.
2. Process as claimed in claim 1, wherein the compound of formula
(II) is prepared by condensing, in the presence of suitable
sylinizing agent, an amide of formula (VIII) ##STR31## where
P.sup.G is a protecting group for the guanidino function, said
compound of formula (VIII) being prepared by forming the amide
group from the corresponding amino acid suitably protected both at
the guanidino group and at the amino group, then unblocking the
amino group, with the compound of formula (XIV) where R.sup.6 is
--(CH.sub.2).sub.4 --NPH.sup.L.
3. A process for preparing a compound of formula (I) ##STR32##
where R is hydrogen or an acyl radical, and R.sup.1 is an
--OP.sup.2 group or an ##STR33## group where R.sup.2 is a hydrogen
atom or a C.sub.1 -C.sub.6 linear or branched chain alkyl radical,
a C.sub.3 -C.sub.6 linear or branched chain alkenyl or alkynyl
radical, or a C.sub.7 -C.sub.12 aryl-alkyl or alkyl-aryl radical,
and R.sup.3 is a hydrogen atom or a hydroxyl group,
a) by condensing a compound of formula (II) ##STR34## where P.sup.L
indicates a protecting group for the side chain amino function, and
P.sup.G indicates a suitable protecting group for the guanidino
function: the compound of formula (II) being prepared by
condensing, in the presence of suitable sylanizing agent, an amide
of formula (VIII) ##STR35## where P.sup.G is a protecting group for
the guanidino function, said compound of formula (VIII) being
prepared by forming the amide from the corresponding amino acid
suitably protected both at the guanidino group and at the amino
group, then unblocking the amino group, with a compound of formula
(XIV) ##STR36## where R.sup.6 is --(CH.sub.2).sub.4 --NHP.sup.L
with a compound of formula (III) ##STR37## where R.sup.4 is a
--OR.sup.2 group in which R.sup.2 is defined as above, an --OP
group where P is a protecting group for the carboxyl function, or a
group of formula (IV) ##STR38## where P is as heretofore defined,
and R.sup.5 is a hydrogen atom or --OP.sup.I group where P.sup.I is
a protecting group for the hydroxyl function of the tyrosine,
wherein the compound of formula (III) is prepared by condensing an
amide of formula (X) ##STR39## where P is a protecting group for
the carboxyl function with a compound of formula (XI) ##STR40##
where R.sup.4 is as above defined, then converting the amide to
primary amine with TIB, said compound of formula (XI) being
prepared by condensing the hemiester of the 2-substituted malonic
acid of formula (XII) ##STR41## and a compound of formula (XIII)
##STR42## where P and R.sup.5 are as above defined. b) followed by
conversion of the terminal amino group of the obtained of formula
(V) ##STR43## into a primary amino group by treatment with
1,1-bis-trifluoroacetoxy-iodobenzene to give an intermediate of
formula (VI) ##STR44## c) followed by possible acylation of the
terminal amino function formed, and the removal of the protecting
groups.
4. A process for preparing a compound of formula (I) ##STR45##
where R is hydrogen and R.sup.1 is an --OP.sup.2 group or an
##STR46## group where R.sup.2 is a hydrogen atom or a C.sub.1
-C.sub.6 linear or branched chain alkyl radical, a C.sub.3 -C.sub.6
linear or branched chain alkenyl or alkynyl radical, or a C.sub.7
-C.sub.12 aryl-alkyl or alkyl-aryl radical, and R.sup.3 is a
hydrogen atom or a hydroxyl group,
a) by condensing a compound of formula (II) ##STR47## where P.sup.L
indicates a protecting group for the side chain amino function, and
P.sup.G indicates a suitable protecting group for the guanidino
function, with a compound of formula (III) ##STR48## where R.sup.4
is a --OR.sup.2 group in which R.sup.2 is defined as above, an --OP
group where P is a protecting group for the carboxyl function, or a
group of formula (IV) ##STR49## where P is as heretofore defined,
and R.sup.5 is a hydrogen atom or --OP.sup.I group where P.sup.I is
a protecting group for the hydroxyl function of the tyrosine,
wherein the compound of formula (III) is prepared by condensing, in
the presence of a suitable sylanizing agent, an amide of formula
(X), said compound (X) obtained from Daspartic acid, ##STR50##
where P is a protecting group for the carbonyl function, with a
compound of formula (XI) ##STR51## where R.sup.4 is a group of
formula (IV), then converting the amide to primary amine with TIB,
said compound of formula (XI) being prepared by condensing, in
presence of a suitable sylanizing agent, the compound of formula
(XIV) ##STR52## where R.sup.6 is --CH(CH.sub.3)CH.sub.3 with a
compound of formula (XIII) ##STR53## where P and R.sup.5 are
defined as above. b) wherein the compound of formula (V) obtained
by condensing the fragments (II) and (III) ##STR54## id deprotected
to give a compound of formula (VII) ##STR55## which is then
converted into the required product by treatment with
1,1-bis-trifluoroacetoxy-iodiobenzene.
5. Process as claimed in claim 4, wherein the compound of formula
(II) is prepared by condensing, in the presence of suitable
sylinizing agent, an amide of formula (VIII) ##STR56## where
P.sup.G is a protecting group for the guanidino function, said
compound of formula (VIII) prepared by forming the amide group from
the corresponding amino acid sutably protected both at the
guanidino group and at the amino group, then unblocking the amino
group, with the compound of formula (XIV) where R.sup.6 is
--(CH.sub.2).sub.4 --NPH.sup.L.
6. A process for preparing a compound of formula (I) ##STR57##
where R is hydrogen, and R.sup.1 is an --OP.sup.2 group or an
##STR58## group where R.sup.2 is a hydrogen atom or a C.sub.1
-C.sub.6 linear branched chain alkyl radical, a C.sub.3 -C.sub.6
linear or branched chain alkenyl or alkynyl radical, or a C.sub.7
-C.sub.12 aryl-alkyl or alkyl-aryl radical, and R.sup.3 is a
hydrogen atom or a hydroxyl group,
a) by condensing a compound of formula (II) ##STR59## where P.sup.L
indicates a protecting group for the side chain amino function, and
P.sup.G indicates a suitable protecting group for the guanidino
function, with the compound of formula (II) being prepared by
condensing, in the presence of suitable sylanizing agent, an amide
of formula (VIII) ##STR60## where P.sup.G is a protecting group for
the guanidino function, with a compound of formula (XIV) ##STR61##
where R.sup.6 is --(CH.sub.2).sub.4 --NHP.sup.L said compound of
formula (VIII) being prepared by forming the amide group from the
corresponding amino acid suitably protected both at the guanidino
group and at the amino group, then unblocking the amino group, with
the compound of formula (XIV) with a compound of formula (III)
##STR62## where R.sup.4 is a --OR.sup.2 group in which R.sup.2 is
defined as above, an --OP group where P is a protecting group for
the carboxyl function, or a group of formula (IV) ##STR63## where P
is as heretofore defined, and R.sup.5 is a hydrogen atom or
--OP.sup.I group where P.sup.I is a protecting group for the
hydroxyl function of the tyrosine, wherein the compound of formula
(III) is prepared by condensing an amide of formula (X) ##STR64##
where P is a protecting group for the carboxyl function with a
compound of formula (XI) ##STR65## where R.sup.4 is as above
defined, then converting the amide to primary amine with TIB, said
compound of formula (XI) being prepared by condensing the hemiester
of the 2-substituted malonic acid of formula (XII) ##STR66## and a
compound of formula (XIII) ##STR67## where P and R.sup.5 are as
above defined, b) wherein the compound of formula (V) obtained by
condensing the fragments (II) and (III) ##STR68## id deprotected to
give a compound of formula (VII) ##STR69## which is then converted
into the required product by treatment with
1,1-bis-trifluoroacetoxy-iodiobenzene.
7. Intermediates of formulas (V), (VI) and (VII) ##STR70## where
P.sup.L indicates a protecting group for the side chain amino
function, P.sup.G indicates a suitable protecting group for the
guanidino function, R.sup.4 is --OR.sup.2 group in which R.sup.2 is
as defined in claim 1, an --OP group where P is a protecting group
for the carboxyl function, or a group of formula (IV) ##STR71##
where P is as heretofore defined, and R.sup.5 is a hydrogen atom or
a --OP.sup.I group in which P.sup.I is a protecting group for the
hydroxyl function of the tyrosine.
8. Intermediate of formula (V), (VI) or (VII) of claim 7, where
P.sup.L indicates a tert-butoxycarbonyl or tert-amyloxycarbonyl
group, P.sup.G indicates a benzenesulphonyl group which may be
substituted, an --OP group where P is a tert-butyl, tert-amyl,
benzyl or substituted benzyl, or a group of formula (IV) ##STR72##
where P is as heretofore defined, and R.sup.5 is a hydrogen atom or
a --OP.sup.I group in which P.sup.I is a tert-butyl or tert-amyl
group.
Description
This invention relates to new analogues of thymopentin (TP5) and
its tetrapeptide fragment (TP5.sup.1-4), containing in the peptide
chain two retro-inverted bonds, the process for preparing these new
compounds and their use in pharmaceutical formulations. In
particular, the present invention firstly relates to analogues of
thymopentin and its tetrapeptide fragment (TP5.sup.1-4) having the
following general formula (I) ##STR3## where R is hydrogen or an
acyl radical, and R.sup.1 is an --OR.sup.2 group or an ##STR4##
group where R.sup.2 is a hydrogen atom or a C.sub.1 -C.sub.6 linear
or branched chain alkyl radical, a C.sub.3 -C.sub.6 linear or
branched chain alkenyl or alkynyl radical, or a C.sub.7 -C.sub.12
aryl-alkyl or alkyl-aryl radical, and R.sup.3 is a hydrogen atom or
a hydroxyl group, and the corresponding pharmaceutically acceptable
salts of acid or basic addition.
The new compounds of the present invention as heretofore defined by
their distinguishing chemical structural formula can be indicated
more concisely using the symbols internationally recognised in the
peptide field, as:
where R is hydrogen or an acyl radical, and R.sup.1 is an
--OR.sup.2 group or a --Tyr--OR.sup.2 or --Phe--OR.sup.2 group
where R.sup.2 is a hydrogen atom or a radical as heretofore
defined.
In this latter formula, gArg indicates a geminal diamino residue
derivable from the amino acid arginine by replacing the carboxyl
with an amino group, and mLys indicates a malonyl residue
substituted in position 2- with the side chain of the amino acid
lysine. Likewise, gAsp indicates a geminal diamino residue
derivable from aspartic acid by replacing the carboxyl with an
amino group, and mVal indicates a malonyl residue substituted in
position 2- with the side chain of the amino acid valine. Finally,
Tyr and Phe represent the tyrosine and phenylalanine residues
respectively.
For the purposes of the present invention the term "acyl radical"
signifies those acyl radicals deriving from C.sub.1 -C.sub.6 linear
or branched chain alkanoic acids such as formyl, acetyl, propionyl,
succinoyl etc., and those aromatic acyl radicals deriving from
benzoic and substituted benzoic acid such as benzoyl,
4-nitrobenzoyl, 2,3,4-trimethoxybenzoyl etc.
As used herein, the term "pharmaceutically acceptable salts"
indicates those salts of acid or basic addition in which,
respectively, the anion or cation is relatively non-toxic and
innocuous when the compounds are administered in the form of salts
of addition at therapeutically effective doses, so that any side
effects of the anions or cations do not cancel the beneficial
effects of the active principle.
Acids able to form pharmaceutically acceptable salts of acid
addition with the compounds of formula (I) include for example
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulphuric acid etc., organic carboxylic acids such
as formic acid, acetic acid, propionic acid, lactic acid, malonic
acid, succinic acid etc., and organic sulphonic acids such as
methanesulphonic acid and naphthalenesulphonic acid. Bases able to
form pharmaceutically acceptable salts with the compounds of
formula (I) include for example mineral bases such as sodium
hydroxide, potassium hydroxide, ammonium hydroxide etc., and
organic bases such as triethylamine, triethanolamine etc. These
salts of addition can either be obtained directly via the synthesis
process for the new retro-inverso peptides, or be prepared by
conventional methods starting from compounds of formula (I), in the
form of free bases or acids, by treating with one or more
equivalents of the chosen acid or base.
If desired, a given salt of acid addition can be converted into
another by treating the former with a suitable ion exchange resin
as described for example by R. A. Boissonas et al. in Helv. Chim.
Acta, 43, 1349 (1960).
A preferred group of compounds of the present invention comprises
those compounds of formula (I) in which R represents a hydrogen
atom, or a metabolically labile acyl radical, or an acyl radical of
which the bond to the amino group is rapidly cleaved during the
initial stages of the metabolic path of the product and which does
not have toxic effects or controindications in therapy when used in
a concentration in which the compound of formula (I) provides the
desired pharmacological effect, R.sup.1 is as heretofore defined
and R.sup.2 is hydrogen.
A more preferred group of compounds comprises those compounds of
formula (I) in which R is a hydrogen atom, R.sup.1 is as heretofore
defined and R.sup.2 is hydrogen.
A still more preferred group of compounds comprises those compounds
of formula (I) in which R is a hydrogen atom and R.sup.1 is a
##STR5## group where R.sup.2 is a hydrogen atom and R.sup.3 is as
heretofore defined. The compounds of the present invention have
demonstrated considerable activity on the immune system.
In the last fifteen years G. Goldstein and his group have studied
in detail the biological action and the possible pharmacological
use of a polypeptide hormone secreted by the epithelial cells of
the thymus, namely thymopoietin [G. Goldstein, Nature, 247, 11
(1974); D. H. Schlesinger et al., Cell, 5, 361 (1975); T. Audhya et
al. Biochemistry, 20, 6195 (1981)], the primary sequence of which
comprises 49 amino acids.
Thymopoietin performs various regulatory actions in the body,
influencing neuromuscular transmission [G. Goldstein, Lancet, 2,
119 (1968)], differentiation between T and B lymphocytes [M. P.
Scheid et al., J. Exp. Med., 147, 1727 (1978)], and the immune
response [C. Y. Lau et al., J. Immunol., 125, 1634 (1980)].
Structure-activity studies have shown that the entire 49 amino acid
sequence of thymopoietin is not required for the biological
activity, in that the pentapeptide H-Arg-Lys-Asp-Val-Tyr-OH
(Thymopentin, TP5) corresponding to the amino acid sequence 32-36
possesses the entire biological activity of the natural hormone
both in vitro and in vivo [G. Goldstein et al., Science, 204, 1309
(1979)].
Thymopentin has already been clinically introduced, both in the
treatment of diseases of autoimmune origin such as rheumatoid
arthritis, and as a stimulant for the body defence system in the
treatment for example of primary immunodeficiency, due to the
absence or incomplete development of the thymus and consequent
alteration in the maturation of the T lymphocytes and of the acute
or recurrent viruses, or as an aid in vaccination.
Setting up the therapeutic protocol is made critical by the
difficulty in determining the effective dose, in that the
pharmacological effect can vary considerably according to the
method and duration of administration [T. Audhya et al., Surv.
Immunol. Res., 4th suppl., 1, 17 (1985); and T. Audhya et al., Int.
J. Peptide Protein Res., 22, 568 (1983)].
The limiting example is that reported by Bolla et al. in Int. J.
Clin. Pharm. Res., IV(6), 431 (1984) according to which the
stimulating effect on the production of antibodies by TP5 when
administered subcutaneously is totally suppressed if the same dose
is administered intravenously. A possible cause of this is the
short half-life of TP5 in the plasma. In this respect, TP5 is
rapidly degraded in the plasma (t.sub.1/2 =1.5 min) by the action
of various proteases [T. P. Tischio et al., Int. J. Peptide Protein
Res., 14, 479 (1979)].
Intense research has been carried out in recent years for the
purpose of obtaining TP5 analogues with increased resistance to
proteases. Mention can be made for example of European patent
application EP-A-135722 and U.S. Pat. No. 4,505,853 relative to
thymopentin analogues obtained by suitably varying the individual
amino acids within the peptide sequence.
Again for this purpose, analogues of thymopentin and of the
tripeptide fragment (TP.sup.1-3) have recently been synthesized in
which the most labile of the bonds in the peptide chain, i.e. the
bond between the arginine residue and the lysine residue [see J. P.
Tischio et al., Int. J. Peptide Protein Res. 14, (1979), pp
479-484, and in particular FIG. 4] has been suitably
retro-inverted. The compounds obtained in this manner, which have
maintained if not improved the immunomodulating activity of
thymopentin and its fragment, have proved much more stable than the
corresponding non-retroinverted compounds towards peptidase. In
particular, for example whereas the half life for thymopentin in
heparinized human plasma is 1.5 minutes, that of the analogue
retro-inverted at the Arg-Lys bond under the same experimental
conditions is 22 minutes. Although this is a considerable
improvement, it is also apparent that if compounds with the
pharmacological possibilities of thymopentin but with increasingly
long half lives were available, the therapeutic use of thymopentin
could be rationalized to the maximum extent.
It has now been surprisingly found that by inverting not only the
bond between the arginine and the lysine but also the bond between
the aspartic acid and the valine, products are obtained which
although maintaining the immunomodulating activity spectrum of
thymopentin, are practically refractory to enzymatic demolition.
For example, the thymopentin analogue in which the two Arg-Lys and
Asp-Val bonds have been retro-inverted (the compound of Example 1),
when tested for stability to enzymatic hydrolysis in heparinized
human plasma in parallel with thymopentin and with thymopentin
retro-inverted only at the Arg-Lys bond, showed a half life of more
than 5 hours. This result is even more surprising considering that
in the natural peptide the cleavage of the Asp-Val bond is of
little importance (see J. P. Tischio et al., the aforesaid
reference and in particular FIG. 4 which shows the possible
degradation scheme of thymopentin in the plasma, and where Asp-Val
cleavage never appears), whereas evidently this cleavage becomes
crucial in the retro-inverted analogue.
The compounds of the present invention are conveniently prepared by
condensing a fragment of formula (II) ##STR6## where P.sup.L
indicates a protecting group for the side chain amino function, and
P.sup.G indicates a suitable protecting group for the guanidino
function, with a fragment of formula (III) ##STR7## where R.sup.4
is a --OR.sup.2 group in which R.sup.2 is as heretofore defined, a
--OP group where P is a protecting group for the carboxyl function,
or a group of formula (IV) ##STR8## where P is as heretofore
defined, and R.sup.5 is a hydrogen atom or a --OP.sup.I group where
P.sup.I is a protecting group for the hydroxyl function of the
tyrosine, followed by conversion of the terminal amido group of the
obtained intermediate of formula (V) ##STR9## into a primary amino
group by treatment with 1,1-bis-trifluoroacetoxy-iodobenzene (TIB)
to give an intermediate of formula (VI) ##STR10## followed by
possible acylation of the terminal amino function formed, and the
removal of the protecting groups. If a compound of formula (I) is
required in which R is hydrogen and R.sup.1 is a --OR.sup.2 group,
the order of the steps involved in conversion of the amide to
primary amine and the deprotection can be conveniently reversed.
This is however not possible if a compound of formula (I) is to be
obtained in which R.sup.1 represents the --Tyr--OR.sup.2 group in
that the phenolic hydroxyl of the tyrosine is extremely sensitive
to TIB and must be protected during the amide/amine conversion
step.
Thus if a compound of formula (I) is required in which R.sup.1 is a
--OR.sup.2 group, unblocking the protecting groups before the
amide/amine conversion by the TIB will lead to an intermediate of
formula (VII) ##STR11## which is reacted with the
1,1-bis-trifluoroacetoxy-iodobenzene to give the required
retro-inverso peptide of formula (I). Consequently, the present
invention also provides the intermediate compounds of formulas (V),
(VI) and (VII) which are obtained during the synthesis of the
pharmacologically active products of formula (I).
In greater detail, the first step of the aforesaid reaction scheme
can be conveniently effected by any of the methods known in the
literature for peptide synthesis. Excellent results in terms of
yield and product purity have been obtained using a carbodiimide
such as dicyclohexylcarbodiimide or diisopropylcarbodiimide and
1-hydroxybenzotriazole. Specifically the reaction is effected by
adding a slight excess of the 1-hydroxybenzotriazole to a solution
of the acid of formula (II) maintained at low temperature, followed
by adding the dicyclohexyl- or diisopropyl-carbodiimide, and then
the reaction partner of formula (III).
For this condensation reaction, which can be conveniently carried
out at ambient temperature, conventional aprotic polar organic
solvents able to solubilize the reactants and not interfere
negatively with the progress of the reaction are used. Selected
solvents are dimethylformamide, acetonitrile and
dimethylsulphoxide, possibly mixed with less polar solvents such as
halogenated aliphatic hydrocarbons, eg. methylene chloride,
dichloroethane etc.
Protecting groups which can be conveniently used in this synthesis
are the conventional groups known in the literature and commonly
used in classical peptide synthesis. In particular, P.sup.L is
preferably a tert-butoxy-carbonyl group or benzyloxycarbonyl group,
possibly nitro- or halo-substituted; P.sup.G is preferably a
benzenesulphonyl group variously substituted, such as an
alkylbenzenesulphonyl group, eg. toluenesulphonyl, or an
alkylalkoxybenzenesulphonyl group, eg.
4-methoxy-2,3,6-trimethylbenzensulphonyl; P.sup.I is preferably a
tert-butyl or tert-amyl group in that such groups have proved
stable to the action of TIB; and finally P can be any protecting
group for the terminal carboxyl functions, such as an alkyl group,
eg. tert-butyl or tert-amyl, or an arylalkyl group, eg. benzyl or
substituted benzyl. When the condensation reaction, the progress of
which can be easily followed by TLC, is complete, the product
obtained is recovered by conventional methods.
In particular, according to a preferred aspect, if using a
carbodiimide as coupling agent the product is recovered by
filtering off the urea which forms, evaporating the solvent,
washing the residue or a solution of the residue in a suitable
organic solvent with weakly basic and weakly acid solutions, and
finally purifying the product by crystallization or
chromatography.
The product obtained in this manner is then reacted with
1,1-bistrifluoroacetoxy-iodobenzene (TIB) by the method described
in U.S. patent application Ser. No. 448,831, by which the amide
substrate is reacted with a slight excess of TIB in an aqueous
mixture of an inert solvent such as dimethylformaldehyde,
acetonitrile, etc. The reaction is effected by bubbling an inert
gas, typically nitrogen, into the reaction mixture and monitoring
its progress by TLC. When complete conversion of the amide into
amine is noted, the organic solvent is removed and the product
easily recovered by lyophilization. If desired the product obtained
in this manner can then be acylated using active esters of the acid
R-OH, such as the p-nitro-phenyl ester, the 2,4,5-trichloro-phenyl
ester etc. The deprotection step is then effected by methods known
in this field. In general, if conventional protecting groups are
used, such as tert-butyl or tert-amyl for the carboxyl and hydroxyl
of the tyrosine, the tertbutoxycarbonyl or benzyloxycarbonyl groups
for the amino group of the lysine, and the benzenesulphonyl groups
for the guanidino group of the arginine, these are conveniently
removed by acidolysis in an acid medium such as with dilute
hydrochloric acid in acetic acid, with trifluoroacetic acid or with
a mixture of trifluoroacetic acid and trifluoromethanesulphonic
acid, in the presence of a small percentage of ethanedithiol,
anisole, thioanisole or resorcinol used as scavengers to trap the
carbocations which form. After termination of the deprotection step
the desired product of formula (I) is recovered either as such or
as the salt of addition, and is then purified by conventional
methods.
If a compound of formula (I) is required in which R is a hydrogen
atom and R.sup.1 is a --OR.sup.2 group, as stated, the order of the
amide/amine conversion and deprotection steps is preferably
reversed.
The general methods for their implementation are however
unchanged.
The obtained compounds of formula (I) are tested for homogeneity by
TLC and HPLC, and for purity by amino acid analysis and NMR. The
starting compounds of formula (II) and (III) can be easily prepared
either from commercially available compounds or from compounds
prepared for the purpose by known methods in the field of peptide
and organic synthesis.
In particular, the fragment of formula (II) can be conveniently
prepared from an amide of formula (VIII) ##STR12## where P.sup.G is
a protecting group for the guanidino function, and a hemiester of
the 2-substituted malonic acid of formula (IX) ##STR13## where
P.sup.L is a suitable protecting group for the amino function,
following the known peptide condensation methods.
The compound of formula (VIII) is prepared by forming the amide
from the corresponding amino acid suitably protected both at the
guanidino group and at the amino group, then unblocking the amino
group, while the compound of formula (IX) is prepared from diethyl
malonate by inserting the substituting group in the 2- position
followed by partial hydrolysis. The fragment of formula (III) is
also conveniently prepared by condensing an amide of formula (X)
##STR14## where P is a protecting group for the carboxyl function,
with a compound of formula (XI) ##STR15## where R.sup.4 is as
heretofore defined, then converting the amide to primary amine with
TIB.
The compound of formula (XI) where R.sup.4 is a group of formula
(IV) can be prepared, in accordance with schemes known in the
literature, from the hemiester of the 2-substituted malonic acid of
formula (XII) ##STR16## and a compound of formula (XIII) ##STR17##
where P and R.sup.5 have the aforesaid meanings.
Alternatively, as illustrated in the patent application filed on
the same date by the present applicant, instead of using a malonic
acid derivative of formula (IX) or (XII), a compound of formula
(XIV) ##STR18## can be used where R.sup.6 is a --(CH.sub.2).sub.4
--NHP.sup.L or --CH(CH.sub.3)CH.sub.3 group respectively, and
effecting the condensation with the amide of formula (VIII) or (X),
or with the compound of formula (XIII), in the presence of a
suitable sylanizing agent.
The compounds of the present invention can exist in four isomeric
forms.
In this respect, in the structural formula (I) there are at least
four asymmetric carbon atoms, but the absolute configuration of the
terminal amino acid (Tyr or Phe) is the L- (this configuration
being obtained using in the synthesis the suitable L-tyrosine or
L-phenylalanine derivatives), and the absolute configuration of the
two gem-diamino carbons is also fixed in that the fragment (VIII)
is obtained from D-arginine and the fragment (X) is obtained from
D-aspartic acid. Finally, the carbon atoms of the malonyl residues
can be of R or S configuration. Consequently the compounds of
formula (I) can be obtained and used in optically active form or in
the form of an isomer mixture. When the synthesis process for the
products of formula (I) leads to a mixture, this can if desired be
resolved into the individual isomers by known resolution
methods.
As already anticipated, the thymopentin with two retro-inverted
peptide bonds has proved extremely stable to the action of
plasmatic peptidase. In particular the lability of
[gArg.sup.1,(R,S)mLys.sup.2,gAsp.sup.3,(R,S)mVal.sup.4 ]TP5 to
enzymatic hydrolysis has been tested in parallel with both TP5 and
[gArg.sup.1,(R,S)mLys.sup.2 ]TP5, using heparinized human plasma
and incubating the three peptides separately at a concentration of
about 30 nmoles/ml of plasma. The incubations were done at
37.degree. C., and the withdrawn samples of the plasmatic mixture,
of 100 .mu.l each, were blocked at the various times by adding
trifluoroacetic acid to the extent of 10%, followed by centrifuging
at 10,000g for 5 minutes. An aliquot of the supernatant was
chromatographed under conditions suitable for showing the change in
the peptide concentration at the various times. The enzymatic data
obtained enabled the half life to be calculated, i.e. the time of
incubation at 37.degree. C. required for degrading 50% of the
tested peptide, this being shown in the following Table I.
TABLE I ______________________________________ Stability towards
enzymatic hydrolysis in human plasma (t/2 in minutes)
______________________________________ TP5 .apprxeq.1.5
[gArg.sup.1,(R,S)mLys.sup.2 ]TP5 22
[gArg.sup.1,(R,S)mLys.sup.2,gAsp.sup.3,(R,S)mVal.sup.4 ]TP5 >300
______________________________________
This considerably increased stability of the bi-retroinverted
analogues relative to TP5 and the mono-retroinverted analogue has
also been demonstrated using isolated enzymes (leucinaminopeptidase
and carboxypeptidase).
The compound of Formula 1 has also shown considerable
immunostimulating activity (greater than that of TP5 or of the
mono-retro-inverted analogue) at extremely low doses. The
immunostimulating activity of
[gArg.sup.1,(R,S)mLys.sup.2,gAsp.sup.3, (R,S)mVal.sup.4 ]TP5 was
tested in vivo compared with that of TP5 and of
[gArg.sup.1,(R,S)mLys.sup.2 ]TP5.
Specifically, hemolysis plates (PFC) were used for the test, which
was conducted by the method described by Jerne and Nordin. The test
was carried out by intravenously administering to C3H/HeNCr1Br
inbred male mice (Calco-Italia) of 10-12 weeks and about 25 g body
weight an apyrogenic saline solution (0.2 ml) containing
1-2.times.10.sup.8 red blood cells from sheep (SRBC). After two
hours, groups each containing 3 mice were treated orally
(intragastric tube) with 0.2 ml of saline solution (control) or
with 0.2 ml of saline solution containing 1 ng of peptide. After 4
days the mice were killed and the spleen withdrawn and dissociated
mechanically to separate the lymphocytes. The thus isolated
lymphocytes were washed with minimum essential medium (MEM)
(3.times.15 ml) containing Earle's salts and then resuspended in
the same medium (1 ml) at a final concentration of 150,000 cells.
Each cell suspension (0.1 ml) was then diluted 1:100 with MEM, 100
.mu.l of each dilution being doubly added to microplate wells
containing 25 .mu.l of MEM, 25 .mu.l of a 10% solution of SRBC
antigens and 25 .mu.l of guinea pig complement at a final dilution
of 1:64. The entire suspension was immediately transferred by
capillarity from each well to superimposed slides. The slides,
closed at their edges with paraffin, were then incubated in an
environment temperature controlled at 37.degree. C. for one
hour.
At the end of this period the direct hemolysis plaques indicating
the number of lymphocytes which secrete antibodies (PFC) are
counted by a light contrast viewer. The antibodies displayed are of
the IgM class, i.e. of primary antibody response. The results,
expressed in % relative to the control, are given in Table II
TABLE II ______________________________________ Plaque formation
Compound % control ______________________________________ TP5 104
TP5 (RI.sub.1-2) 112 TP5 (RI.sub.1-2)(RI.sub.3-4) 219
______________________________________
In Table II, as elsewhere in this patent, "RI" indicates
"retro-inverso". TP5 (RI.sub.1-2) consequently indicates the TP5
analogue retro-inverted at the 1-2 peptide bond, and TP5
(RI.sub.1-2)(RI.sub.3-4) indicates the thymopentin analogue
retro-inverted both at the 1-2 bond and at the 3-4 bond.
Because of their biological characteristics, the peptides of the
present invention are able to interfere with the body immune
response, to substantially stimulate it when deficient. The
compounds of the present invention are therefore therapeutically
useful in treating a series of pathological states related to
immunodeficiency. These include for example Di George's syndrome,
characterised by a congenital absence of the thymus, or chronic or
long-duration viral, fungal or microplasmatic infections. The
present invention therefore further provides pharmaceutical
compositions containing a therapeutically effective quantity of one
or more of the compounds of formula (I).
For therapeutic use as immunostimulators or immunomodulators, the
compounds of the present invention can be conveniently administered
parenterally, orally or sub-lingually. The formulations containing
the new compounds can be prepared in the conventional manner by
combining the active principle with an inert vehicle and possibly
with suitably chosen conventional additives.
For oral or sub-lingual use, the compounds of the present invention
can be administered in the form of tablets, capsules, drops,
elixirs etc., prepared using conventional vehicles/excipients such
as starch, sugar, water, alcohol etc. and possibly containing
flavourings, stabilizers, preservatives, lubricants etc. For
parenteral use, the chosen vehicle is sterile water for injections.
Additives can be added in the known manner. The therapeutically
effective daily dose varies according to the subject to be treated
(weight, age and condition) and the method of administration.
However, generally the compounds of the invention are active when
administered at a daily dose of between 2 and 200 ng/kg. The
pharmaceutical formulations of the present invention therefore
contain the compounds of formula (I) in a quantity suitable to
ensure correct daily dosage within the aforesaid range.
The following examples describe in detail some compounds
representative of the present invention and their method of
synthesis.
EXAMPLE 1
Synthesis of [gArg.sup.1,(R,S)mLys.sup.2,gAsp.sup.3,(R,S)mVal.sup.4
]TP5 acetate
(the acetic acid addition salt of the compound of formula (I)
where: R=H. ##STR19##
1) (M)Val (2,2-dimethyl-5-isopropyl-1,3-dioxane-4,6-dione) A
solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum's acid)
(14.4 g, 100 mmoles) in acetone (50 ml) containing pyridine (0.5
ml, 5 mmoles) and glacial acetic acid (0.3 ml, 5 mmoles) is stirred
at ambient temperature for 6 hours, then evaporated to dryness. The
residue is taken up in methanol (MeOH) (150 ml) and NaBH.sub.4
(4.16 g, 110 mmoles) is added. After 15 minutes the MeOH volume is
reduced to about 50 ml. H.sub.2 O (50 ml) is added and the solution
adjusted to pH 3 by adding 3N HCl. The white precipitate obtained
is recovered by filtration and dried in an oven (11.6 g, yield
63%). M.P. 98.degree.-99.degree. C. .sup.1 H-NMR analysis confirms
the assigned structure. chromatographic analysis carried out under
the following conditions shows no trace of impurities:
Column: HiBar, Lichrosorb.RTM. RP-18 (10.mu.)
Eluents: gradient from 0 to 40% of solution B in A within the first
20 minutes, from 40 to 80% of B in A within the next 10 minutes and
then constant at 80% for a further 5 minutes (B=0.1% TFA in
CH.sub.3 CN; A=0.1% TFA in 10% CH.sub.3 CN in H.sub.2 O)
Flow: 1.5 ml/min
Detection: U.V. 230 nm
Single peak T.sub.R =18.27 min.
2) Fmoc-D-Asp(OBu.sup.t)NH.sub.2 [amide of N.sup.a
-fluorenyl-methoxycarbonyl-D-aspartic acid (.beta.-tert-butyl
ester)]
Some ammonium salt of 1-hydroxy-benzotriazole (HOBt.NH.sub.3) (3.21
g, 20 mmoles) and a solution of N,N'-dicyclohexylcarbodiimide (DCC)
(4,13 g, 20 mmoles) in N,N-dimethylformamide (DMF) are added to a
solution of N.sup.a -fluorenyl-methoxycarbonyl-D-aspartic acid
(.beta.-tert-butyl ester) [Fmoc-D-Asp(OBu.sup.t)OH] (8.23 g, 20
mmoles) in DMF (60 ml), cooled to 0.degree. C. and kept stirring in
a nitrogen atmosphere. After about 60 minutes the ice bath is
removed and stirring continued for a further 60 minutes at ambient
temperature, the N,N'-dicyclohexylurea (DCU) which forms is
filtered off and the solvent is removed by evaporation under
reduced pressure. The crude reaction product is taken up in ethyl
acetate (AcOEt) (150 ml), extracted with an aqueous 5% (w/v)
NaHCO.sub.3 solution (3.times.100 ml), and then with an aqueous
saturated solution of NaCl (3.times.100 ml). The organic phase,
dried over MgSO.sub.4, is evaporated to dryness to give a white
gelatinous mass which is triturated with H.sub.2 O and dried in an
oven (7.8 g, yield 95%). M.P.=134.degree.-136.degree. C. .sup.1
H-NMR analysis confirms the assigned structure. Chromatographic
analysis carried out under the conditions of step 1) shows a single
peak with T.sub.R =27.03 min.
3) H-D-Asp(OBu.sup.t)NH.sub.2 [amide of D-aspartic acid
(.beta.-tert-butyl) ester]
The product obtained in the preceding step (7.5 g, 18 mmoles) is
treated for 60 minutes at ambient temperature in a nitrogen
atmosphere, with a 80/20 DMF/diethylamine mixture (100 ml). After
one hour the reaction mixture is evaporated to dryness, the residue
is taken up in AcOEt (100 ml) and extracted with 0.1N HCl
(3.times.70 ml). The aqueous phases are pooled and reduced to a
volume of about 100 ml, then adjusted to pH 9 with a 10% (w/v)
Na.sub.2 CO.sub.3 solution and re-extracted with AcOEt (4.times.50
ml). The pooled phases are dried over MgSO.sub.4 and evaporated
under reduced pressure, to leave an oily residue which is purified
by flash chromatography over silica, eluting with AcOEt and then
with MeOH. 1.45 g of a yellowish oil are obtained (yield 44%).
.sup.1 H-NMR and mass analysis confirm the assigned structure. HPLC
analysis under the conditions of step 1) shows a single peak with
T.sub.R =6.52 min.
4) H-Tyr(Bu.sup.t)OBu.sup.t (tert-butyl ester of
L-tyrosine-O-tertbutylether)
A solution of ammonium formate (2.52 g, 40 mmoles) in MeOH (40 ml)
and some 10% (w/w) Pd/C (3.5 g) are added to a solution of the
tert-butyl ester of
N.alpha.-benzyloxycarbonyl-L-tyrosine-O-tertbutylether
[Z-Tyr(Bu.sup.t)OBu.sup.t ] [prepared by the method described in
New Aspects in Physiological Antitumor Substances, Karger, Basel
(1985) p. 33] (6.92 g, 16 mmoles) in MeOH (60 ml). The resultant
suspension is kept stirring at ambient temperature under a nitrogen
atmosphere for 30 minutes, and then filtered through celite, the
solvent being removed by evaporation under reduced pressure. The
oily residue obtained is taken up in a 10% (w/v) Na.sub.2 CO.sub.3
solution (50 ml) and extracted with AcOEt (4.times.50 ml). The
pooled organic phases are dried over MgSO.sub.4 and the solvent
removed by evaporation under reduced pressure. 3.6 g of a
colourless oily product are obtained (yield 77%). .sup.1 H-NMR
analysis confirms the assigned structure. HPLC analysis under the
conditions of step 1) shows a single peak at T.sub.R =20.90 min.
Tlc (CMA)R.sub.f =0.44.
5) HO-(R,S)mVal-Tyr(Bu.sup.t)OBu.sup.t [tert-butyl ester of
(R,S)-2-isopropyl-malonyl-L-tyrosine-O-tert-butylether]
(M)Val (1.012 g, 5.5 mmoles), N,O-bis-(trimethylsilyl)acetamide
(BSA) (2.45 ml, 10 mmoles) and trimethylsilylchloride (TMSC) (0.634
ml, 5 mmoles) are added in succession to a solution of tert-butyl
ester of L-tyrosine-O-tert-butylether [H-Tyr(Bu.sup.t)OBu.sup.t ]
(1.467 g, 5 mmoles) in tetrahydrofuran (THF) (25 ml). The reaction
is conducted in a nitrogen atmosphere at ambient temperature for 20
hours, then H.sub.2 O (50 ml) is added and the pH adjusted to 4
with citric acid. The reaction mixture is extracted with methylene
chloride (3.times.50 ml), the organic phases are pooled, washed
with water (50 ml), dried over MgSO.sub.4, then evaporated under
reduced pressure. 2.02 g of a colourless oil are obtained (yield
96%).
.sup.1 H-NMR and mass analysis confirm the assigned structure. HPLC
analysis under the conditions of step 1) shows a single peak with
T.sub.R =27.80 min.
6) H.sub.2 N-D-Asp(OBu.sup.t)-(R,S)mVal-Tyr(Bu.sup.t)OBu.sup.t
A solution of HOBt (0.567 g, 3.96 mmoles) in DMF (3 ml) and a
solution of DCC (0.73 g, 3.53 mmoles) in CH.sub.2 Cl.sub.2 (5 ml)
are added to a solution of the product obtained in step 5) (1.49 g,
3.53 mmoles) in CH.sub.2 Cl.sub.2 (20 ml), cooled to 0.degree. C.
and kept stirring in a nitrogen atmosphere. After 60 minutes the
ice bath is removed and stirring continued for a further 60
minutes. A solution is then added of the amide of D-aspartic acid
the side chain carboxyl of which is protected as tert-butyl ester
[H-D-Asp(OBu.sup.t)NH.sub.2 ] (0.62 g, 3.3 mmoles) in CH.sub.2
Cl.sub.2 (7 ml), the reaction being conducted at ambient
temperature for 20 hours.
The DCU which forms is filtered off, the solvent removed by
evaporation under reduced pressure and the crude reaction product
taken up in THF (30 ml). This is then cooled to -15.degree. C. for
2 hours, the DCU precipitate removed by filtration, the solvent
removed by evaporation under reduced pressure and the residue taken
up in AcOET (75 ml). The solution is washed with an aqueous 5%
(w/v) NaHCO.sub.3 solution (3.times.50 ml), then with a saturated
solution of NaCl (3.times.50 ml), with 0.1N HCl (3.times.50 ml) and
finally with H.sub.2 O (3.times.50 ml). The organic phase is dried
over MgSO.sub.4, the solvent evaporated and the residue triturated
with n-hexane (50 ml). A white solid is obtained (1.28 g, yield
65%) with M.P=113.degree.-5.degree. C.
.sup.1 H-NMR and mass analysis confirm the assigned structure. HPLC
analysis under the conditions of step 1) shows a single peak with
T.sub.R =29.21 min.
7) TFA.H-gAsp(OBu.sup.t)-(R,S)mVal-Tyr(Bu.sup.t)OBu.sup.t
A solution of [[bis-(trifluoroacetoxy)iodo]benzene] (TIB) (0.807 g,
1.88 mmoles) in CH.sub.3 CN (5 ml) is added in small portions to a
solution of the compound obtained in the preceding step (1.01 g,
1.706 mmoles) in a 2/1 CH.sub.3 CN/H.sub.2 O mixture (15 ml), kept
stirring in a nitrogen atmosphere. After 3.5 hours at ambient
temperature the solvent is removed and the residue triturated with
a 1/1 ethyl ether/n-hexane mixture (20 ml). A white product (0.815
g, yield 70% is obtained with M.P=143.degree.-145.degree. C.
.sup.1 H-NMR and mass analysis confirm the assigned structure. HPLC
analysis under the conditions of step 1) shows a single peak with
T.sub.R =28.03 min.
8) Fmoc-D-Arg(Mtr)-NH.sub.2 (amide of N.sup.a
-fluorenylmethoxycarbonyl-N.sup.G
-(4-methoxy-2,3,6-trimethyl)benzenesulphonyl-D-arginine
A solution of the ammonium salt of 1-hydroxybenzotriazole
(HOBt.NH.sub.3) (3.0 g, 16.45 mmoles) in DMF (10 ml) and a solution
of DCC (3.38 g, 16.45 mmoles) in DMF (10 ml) are added to a
solution of N.sup.a -fluorenylmethoxycarbonyl-N.sup.G
-(4-methoxy-2,3,6-trimethyl)benzenesulphonyl-D-arginine
[Fmoc-D-Arg(Mtr)-OH] (10 g, 16.45 mmoles) in DMF (50 ml) cooled to
0.degree. C. and kept stirring. After about one hour the
temperature is allowed to rise to ambient and stirring is continued
for a further 60 minutes.
The DCU formed is then removed by filtration and the solvent is
evaporated to obtain an oily residue which is taken up in AcOEt and
washed firstly with an aqueous 5% (w/v) sodium bicarbonate solution
(3.times.50 ml) and then with a saturated sodium chloride solution
(3.times.50 ml).
The organic solution is dried over MgSO.sub.4 and evaporated to
dryness. The solid residue obtained is triturated with ethyl ether
(Et.sub.2 O) (100 ml) to give a colourless powder (9.2 g, yield
93%). The product is characterised by a M.P. of
168.degree.-172.degree. C. HPLC analysis under the conditions of
step 1) shows a single peak with T.sub.R =26.19 min.
9) HCl.H-D-Arg(Mtr)-NH.sub.2 [hydrochloride of N.sup.G
-(4-methoxy-2,3,6-trimethyl)benzenesulphonyl-D-arginine amide]
A suspension of the compound obtained in step 8) (9 g, 15.5 mmoles)
in a 80/20 DMF/diethylamine mixture (100 ml) is left stirring for
one hour. The solvent is then removed by evaporation at reduced
pressure, the residue is taken up in AcOEt (100 ml) and extracted
with an aqueous 0.1N HCl solution (3.times.50 ml). The aqueous
extracts are pooled, washed with further AcOEt (50 ml) and
lyophilized several times to give a colourless flaky product (4.5
g, yield 80%).
The product has no precise melting point.
.sup.1 H-NMR analysis confirms the assigned structure.
HPLC analysis under the conditions of step 1) shows a single peak
at T.sub.R =12.69 min.
10) OEt-mLys(BOC)-OEt [ethyl ester of
2-(N-tert-butoxycarbonyl-4-butylamino)malonic acid]
Sodium metal (0.28 g, 12 mmoles) is dissolved in absolute ethyl
alcohol (EtOH) (9 ml) under a stream of nitrogen. The mixture is
heated to 60.degree. C. and diethyl malonate (3.8 g, 24 mmoles)
added dropwise. N-tert-butoxycarbonyl-4-chloro-butylamine (2.5 g,
12 mmoles) is gradually added at ambient temperature to the
resultant solution. The reaction mixture is then stirred for 2
hours at ambient temperature and for 6 hours at reflux temperature,
and then poured into a 1/1 (v/v) AcOEt/water mixture (100 ml). The
separated organic phase is washed repeatedly with water and dried
over MgSO.sub.4.
The solvent is removed under vacuum at 100.degree. C. leaving an
oily crude product which is purified by reverse phase HPLC using
RP-18 resin and eluting with an aqueous phase modified with
CH.sub.3 Cn (45 vol %). The desired compound is obtained (1.31 g)
as pure product. HPLC analysis under the conditions of step 1)
shows a single peak at T.sub.R =27.08 min.
TLC (n-hexane/AcOEt 70/30) R.sub.f =0.57
11) HO-(R,S)mLys(BOC)-OEt [ethyl hemiester of
2-(N-tert-butoxycarbonyl-4-butylamino)malonic acid]
A solution of KOH (0.66 g, 9.97 mmoles) in EtOH (10 ml) is added
drop by drop over two hours to a suspension of the compound
obtained in the preceding step (3.48 g, 10.5 mmoles) in EtOH (15
ml) kept at 0.degree. C. in a nitrogen atmosphere.
After 16 hours the reaction mixture is taken up in H.sub.2 O (70
ml), reduced to a small volume by evaporation under reduced
pressure and extracted with Et.sub.2 O (3.times.50 ml). The aqueous
phase is then acidified with 1N HCl to pH 3 and further extracted
with AcOEt (3.times. 50 ml). The organic extracts are pooled,
washed with a saturated aqueous NaCl solution and dried over Mg
SO.sub.4. On evaporating the solvent under reduced pressure the
desired product is obtained as a transparent oil (2.64 g, yield
83%). .sup.1 H-NMR analysis confirms the assigned structure.
HPLC analysis under the conditions of step 1) shows a single peak
at T.sub.R =19.58 min.
TLC (CMA) R.sub.f =0.58
12) H.sub.2 N-D-Arg(Mtr)-(R,S)mLys(BOC)-OEt (ethyl ester of (R,S)
[2-(N-tert-butoxycarbonyl)-4-butylamine]-malonyl-N.sup.G
-(4-methoxy-2,3,6-trimethylbenzenesulphonyl)-D-arginine amide)
A solution of HOBt (0.76 g, 6.3 mmoles) in DMF (10 ml) and a
solution of DCC (1 g, 4.85 mmoles) in DMF (5 ml) are added to a
solution of the compound of the preceding step (1.47 g, 4.85
mmoles) in DMF (15 ml) cooled to 0.degree. C. and kept stirred
under a nitrogen atmosphere.
After one hour the temperature is allowed to rise to ambient and
stirring is continued for a further 60 minutes. A solution of the
compound of step 9) (1.86 g, 4.4 mmoles) in DMF (15 ml) containing
triethylamine (TEA) (0.612 ml, 4.4 mmoles) is then added. The
reaction is conducted at ambient temperature for 20 hours, after
which an excess of DCC (0.2 g, 0.97 mmoles) is added and stirring
continued for a further 4 hours.
The DCU which forms is then removed by filtration, the DMF is
removed by evaporation under reduced pressure, the residue is taken
up in THF (20 ml) and left cooled to -15.degree. C. overnight,
after which it is refiltered and evaporated to dryness. The oily
residue obtained is taken up in AcOEt (70 ml) and the organic
solution washed in sequence with an aqueous 5% solution of sodium
bicarbonate (3.times.50 ml), with an aqueous saturated solution of
NaCl (3.times.50 ml), with a 0.1N aqueous HCl solution (3.times.50
ml) and again with the aqueous saturated solution of NaCl
(3.times.50 ml). The organic phase is then dried over MgSO.sub.4
and evaporated. The oily residue obtained is triturated with
hexane, to finally give a finely divided white powder (2.26 g,
yield 76%). The product has a M.P. of 148.degree.-149.degree.
C.
.sup.1 H-NMR and mass analysis confirm the assigned structure. HPLC
analysis under the conditions of step 1) shows a single peak at
T.sub.R =23.38 min.
TLC (CMA) R.sub.f =0.4
13) H.sub.2 N-D-Arg(Mtr)-(R,S)mLys(BOC)-OH [N.sup.a
-(R,S)-malonyl-2-(N-butoxycarbonyl)-4-butylamine)-N.sup.G
-(4-methoxy-2,3,6-trimethylbenzenesulphonyl)-D-arginine amide)
A solution of KOH (0.204 g, 3.63 mmoles) in absolute EtOH (5 ml) is
added drop by drop over a period of 11/2 hours to a solution of the
compound obtained in the preceding step (2.21 g, 3.3 mmoles) in
absolute EtOH (20 ml) cooled to 0.degree. C. and kept under
nitrogen. After 16 hours the reaction mixture is diluted with water
(50 ml), evaporated to a small volume and extracted with Et.sub.2 O
(3.times.50 ml). The aqueous phase is acidified with 0.1N HCl to pH
3 and then further extracted with AcOEt (3.times.50 ml). The
organic extracts are pooled, dried over MgSO.sub.4 and evaporated.
An oily residue is obtained (1.97 g, yield 93%), the structure of
which is confirmed by .sup.1 H-NMR and mass analysis. HPLC analysis
conducted by the standard procedure of step 1) shows a double peak
due to the pair of diastereoisomers at T.sub.R =20.02 and 20.52
min. TLC (CMA) R.sub.f =0.13 and 0.19.
14) H.sub.2
N-D-Arg(Mtr)-(R,S)mLys(BOC)-gAsp(OBu.sup.t)-(R,S)mVal-Tyr(Bu.sup.t)-OBu.su
p.t
A solution of HOBt (35 mg, 0.24 mmoles) in DMF (0.3 ml) and a
solution of DCC (45.4 mg, 0.22 mmoles) in DMF (0.3 ml) are added to
a solution of the compound obtained in the preceding step (141 mg,
0.22 mmoles) in DMF (0.5 ml) after cooling to 0.degree. C., in a
nitrogen atmosphere.
After 60 minutes the temperature is allowed to rise to ambient and
the solution left stirring for a further 60 minutes. A solution of
the compound obtained in step 7) (136 mg, 0.2 mmoles) in DMF (0.9
ml) containing TEA (28 .mu.l, 0.2 mmoles) is then added. After
stirring for 20 hours, the DCU which forms is filtered off and the
filtrate evaporated to dryness. The solid residue obtained is taken
up in AcOEt (7 ml), washed with an aqueous 5% (w/v) NaHCO.sub.3
solution (3.times.5 ml), then with a saturated solution of NaCl
(3.times.5 ml), with 0.1N HCl (3.times.5 ml) and finally with water
(3.times.5 ml). The organic phase is dried over MgSO.sub.4 and
evaporated to dryness. The desired product is obtained (187 mg,
yield 79%). .sup.1 H-NMR and mass analysis confirm the assigned
structure. HPLC analysis displays no trace of impurities and shows
a single peak at T.sub.R =31.84 min.
15)
TFA.H-gArg(Mtr)-(R,S)mLys(BOC)-gAsp(OBu.sup.t)-(R,S)mVal-Tyr(Bu.sup.t)-OBu
.sup.t
The compound of the preceding step (170 mg, 0.14 mmoles) is
dissolved in a mixture containing CH.sub.3 CN (1.5 ml), H.sub.2 O
(1 ml) and DMF (0.5 ml), kept an ambient temperature under a
nitrogen atmosphere. A solution of TIB (66.2 mg, 0.154 mmoles) in
CH.sub.3 CN (0.5 ml) is then added. After 4 hours, the reaction
mixture is evaporated to dryness, and the residue taken up in MeOH
(75 ml) and evaporated to dryness several times. A slightly yellow
coloured vitreous product is obtained (130 mg, yield 73%) the
structure of which is confirmed by mass analysis, and which shows a
purity exceeding 93% on HPLC analysis (T.sub.R =32.03 min).
16) AcOH.H-gArg-(R,S)mLys-gAsp-(R,S)mVal-Tyr-OH
([gArg.sup.1, (R,S)mLys.sup.2, gAsp.sup.3, (R,S)mVal.sup.4 ]TP5
acetate)
The entire product of the preceding step (130 mg, .apprxeq.0.1
mmoles) is treated for 20 minutes at ambient temperature in a
nitrogen atmosphere with a freshly prepared solution containing
trifluoroacetic acid (TEA), trifluoromethanesulphonic acid (TFMSA)
and 1,2-ethanedithiol (EDT) (TFA/TFMSA/EDT 89/1/10) (20 ml). After
20 minutes the reaction mixture is cooled to 0.degree. C. and TEA
(0.3 ml, 1.44 mmoles) is added drop by drop, after which it is
evaporated to dryness under a nitrogen stream. The residue is taken
up in H.sub.2 O (50 ml), extracted with Et.sub.2 O (30 ml) and the
ether phase is counter-extracted with H.sup.2 O (20 ml). The two
aqueous phases are pooled and extracted with Et.sub.2 O (3.times.30
ml) and then evaporated under reduced pressure. The oily residue
obtained is purified by ion exchange chromatography in a column
(15.times.0.9 cm) packed with CM-Sephadex C-25 (2 g) and developed
with a linear gradient in ammonium acetate at pH 4.4, from 0.15 to
0.6M in 8 hours, at a flow of 0.7 ml/min. The fractions containing
the desired product are pooled, concentrated to a small volume
under reduced pressure and lyophilized. 47.2 mg of product are
obtained (yield 64%). .sup.1 H-NMR and mass analysis confirm the
product structure, while HPLC analysis confirms its purity (under
the standard conditions of step 1), showing a single peak at
T.sub.R =8.28 min.
* * * * *